
Cop\i1glii N° 

COPYRIGHT DEPOSIT. 



FOOD AND NUTRITION 

LABORATORY MANUAL 

DEPARTMENT OF HOUSEHOLD SCIENCE 
UNIVERSITY OF ILLINOIS 

Revised Edition 



ISABEL BEVIER, Ph.M. 
SUSANNAH USHER, S.B. 




WHITCOMB & BARROWS 
BOSTON. 1908 



■^Vt-fi 



l,ieftA8Y of C^NiaRESS 



:?>' 



Copyright 1908 

BY 

ISABEL BEVIER and SUSANNAH USHER 



Composition and Electrotyping by 

THOMAS TODD 

14 Beacon Street, Boston, Mass. 



CONTENTS 

Pack 

Introduction 7 

Protein Substances 9 

Fats 27 

Carbohydrates ^;^ 

Milk 45 

Meat 49 

Wheat, Flour, Bread 57 

Digestion of Starch 60 

Digestion of Proteins 62 

Digestion of Fat 66 

Appendix 68 

Bibliography 73 



PREFACE TO SECOND EDITION 

In the second edition of this manual the general plan 
and purpose of the first edition have been retained. The 
results of later investigations have been utilized in regard 
to classification and methods of experimentation. 

The authors wish to acknowledge the valuable assistance 
rendered by Miss Helen Isham, Ph.D., in the revision of 
the experimental work; also their indebtedness to Dr. H. S. 
Grindley, Professor of Animal Chemistry, and Mr. A. D. 
Emmett, Research Assistant in Animal Chemistry in the 
University of Illinois, for valuable suggestions about the 
work in meat and its extractives. 

I. B. 
S. U. 



LABORATORY MANUAL 

INTRODUCTION 

TN the preparation of this laboratory guide the inten- 
-■- tion has not been to devise new and original ex- 
periments, but rather to select from the large body of 
experiments now oiifered in physiological chemistry those 
which in themselves, or in their applications, have a more 
or less direct bearing upon the principles governing the 
selection and preparation of food. 

The task is a somewhat difficult one, because the 
greater number of experiments given in text-books 
of physiological chemistry are designed for students of 
medicine, and therefore put the emphasis upon the 
medical phase of the subject. 

This manual is intended primarily for the use of 
students in the Department of Household Science of the 
University of Illinois. The subject-matter is, therefore, 
arranged with reference to the correlation of the work 
of this department with that of the pure science work 
given in the University. For these students the guide 
is expected to serve two purposes: first, to aid the stu- 
dent to recall and to arrange in an orderly way the 
knowledge gained from other sources; second, to apply 
this knowledge, in so far as possible, to various kinds 
of food problems, and so to serve as an introduction to 
individual work with foods which follows. 

7 



FOOD AND NUTRITION 

PROTEIN SUBSTANCES 

Among food principles proteins assume first impor- 
tance, whether we consider their abundance, the variety 
of forms in which they occur, their part as tissue builders, 
or their decomposition products. Protein is an essential 
constituent of the cell, and not one of the phenomena 
of life is performed without its presence. Different 
varieties of protein exist in both animal and vegetable 
organisms. 

The complexity of protein substances is shown by 
the number of compounds obtained from them, and 
their instability by the ease with which they are broken 
up into other compounds. Neither the molecular struc- 
ture nor the chemical formula of protein is known. The 
elements always found in protein are carbon, hydrogen, 
nitrogen, and oxygen. Many contain sulphur, a few 
phosphorus and iron. 

The percentage composition of some of the more 
important members of the group is within the following 
limits : 

Carbon 51.3-55.0 per cent 

Hydrogen 6.^- 7.3 percent 

Nitrogen I5-5-I9-3 percent 

Oxygen 20.8-23.5 percent 

Sulphur 0.3- 2.2 per cent 

9 



lO Food and Nutrition 

CLASSIFICATION 

Owing to the incompleteness of our knowledge con- 
cerning protein substances, little uniformity exists in 
their classification. The following scheme for classifi- 
cation has been recommended by the joint committees 
of the American Physiological and Bio-Chemical Socie- 
ties on protein nomenclature. 

"Recommendations of the Committee on 

Protein Nomenclature" 

Proteins 

"Since ^ chemical basis for the nomenclature of the 
proteins is at present not possible, it seemed important 
to recommend few changes in the names and definitions 
of generally accepted groups, even though in many cases 
these are not wholly satisfactory. The recommendations 
are as follows: 

"First. The word proteid should be abandoned. 

"Second. The word protein should designate that 
group of substances which consists, so far as at present 
is known, essentially of combinations of a-aniino-acids 
and their derivatives ; e. g., a-amino-acetic acid or glyco- 
coll, a-amino-propionic acid or alanine, phenyl-a-amino 
propionic acid or phenylalanine, guanidine-amino-valeri- 
anic acid or arginine, etc., and are therefore essentially 
polypeptides. 

"Third. That the following terms be used to desig- 
nate the various groups of proteins'': 
I. Simple Proteins. — Protein substances which yield 
only a-amino acids or their derivatives on 
hydrolysis. 



Laboratory Manual ii 

(o) Albumins. — Soluble in pure water and coag- 
ulable by heat ; e. g., egg albumin, serum albu- 
min, lactalbumin, vegetable albumins. 

(b) Globulins. — Insoluble in pure water, but soluble 
in neutral solutions of salts of strong bases with 
strong acids, e. g., serum globulin, ovoglobulin, 
edestin, amandin, and other vegetable globulins. 

(c) Glutelins. — Proteins present in seeds of cereals, 
and insoluble in all neutral solvents. 

(d) Alcohol soluble proteins. — Proteins soluble in 
70 to 80 per cent alcohol, insoluble in water, 
absolute alcohol, and other neutral solvents, 
e. g., zein, gliadin, hordein. 

(e) Albuminoids. — Proteins possessing a similar 
structure to those already mentioned, but char- 
acterized by a pronounced insolubility in all 
neutral solvents, e. g., elastin, collagen, keratin. 

(/) Histones. — Basic proteins which stand between 
protamines and true proteins, e. g., globin, thy- 
mus histone, scombron, etc. 
{g) Protamines. — The simplest natural proteins, 
possessing strong basic properties and forming 
stable salts with strong mineral acids, e. g., 
salmin, sturin, clupein, scombrin. 
II. Conjugated Proteins. — Substances which contain 
the protein molecule united to some other mole- 
cule or molecules otherwise than as a salt, 
(o) Nucleoproteins. — Compounds of one or more 
protein molecules with nucleic acid, e. g., cyto- 
globulin, nucleohistone. 



12 Food and Nutrition 

(b) Glycoproteins. — Compounds of the protein 
molecule with a substance or substances con- 
taining a carbohydrate group other than a 
nucleic acid, e. g., mucine and mucoids ( Osseo- 
mucoid, tendomucoid, ichthulin, helicoprotein, 
etc.). 

{c) Phosphoproteins. — Compounds of the protein 
molecule with some, as yet undefined, phos- 
phorus — containing substances other than a 
nucleic acid or lecithin, e. g., caseinogen, vitellin, 
etc. 

{d) Haemoglobins. — Compounds of the protein 
molecule with haematin, or some similar sub- 
stance, e. g., haemoglobin, haemocyanin. 

(e) Lecithoproteins. — Compounds of the protein 
molecule with lecithins, e. g,, lecithans, phos- 
phatides. 
III. Derived Proteins. 

I. Primary Protein Derivatives. — Derivatives of the 
protein molecule apparently formed through hy- 
drolytic changes which involve only slight alter- 
ation of the protein molecule. 

(a) Proteans. — Insoluble products which apparently 
result from the incipient action of water, very 
dilute acids, or enzymes, e. g., myosan, edestan. 

{b) Metaproteins. — Products of the further action 
of acids and alkalis whereby the molecule is so 
far altered as to form products soluble in very 
weak acids and alkalis, but insoluble in neutral 
fluids, e. g., acid albuminate, alkali albuminate. 



Laboratory Manual 13 

(c) Coagulated proteins. — Insoluble products which 

result from (i) the action of heat on their 

solutions, or (2) the action of alcohol on the 

protein. 

2. Secondary Protein Derivatives. — Products of the 

further hydrolytic cleavage of the protein molecule. 

(a) Proteoses. — Soluble in water, non-coagulable 
by heat, and precipitated by saturating their 
solutions with ammonium, or zinc sulphate, 
e. g., protoproteose, deuteroproteose. 

(b) Peptones. — Soluble in water, non-coagulable 
by heat, but not precipitated by saturating their 
solutions with ammonium sulphate, e. g., anti- 
peptone, amphopeptone. 

(c) Peptides. — Definitely characterized combina- 
tions of two or more amino-acids, the carhoxyl 
group of one being united with the amino group 
of the other with the elimination of a molecule 
of water, e. g., dipeptides, tripeptides, tetrapep- 
tides, pentapeptides, etc. 

For the purposes of this manual the following scheme 
is deemed sufficient : 
I. Proteins. 

I. Simple Proteins. 

(a) Albumins — egg albumin, serum albumin, lacta 

albumin, and vegetable albumins. 
(6) Globulins — serum globulin, ovoglobulin, edes- 

tin, and other vegetable globulins. 
(c) Phospho-proteins (nucleo-albumins) — caseino- 
gen and vitellin. 



14 Food and Nutrition 

2. Derived Proteins. 

(a) Albuminates — acid albuminate and alkali albu- 
minate. 

(&) Proteoses (or albumoses) and peptones — pro- 
toproteose, heteroproteose, and deuteroproteose ; 
amphopeptone and antipeptone. 

(c) Coagulated proteins — fibrin, and the products 
of heat coagulation, etc. 

II. Compound Proteins. 

Include compounds of simple proteins with other 
bodies ; for example, with coloring matter, haemo- 
globin; with carbohydrates, glucoprotein ; with 
nuclein or nucleic acid, nucleoprotein. 

III. Albuminoids. 
Collagen, elastin, gelatin. 

For fuller information on the subject of proteins the 
following authors may be consulted : Hammarsten, Hawk, 
Jackson, Long, Mann, Salkowski. 

COMPOSITION OF PROTEINS 

ExPT. I. Burn a small piece of dry albumin in a 
test-tube. Why does it turn black? On continued heat- 
ing, what is left? Notice the characteristic odor. 

ExPT. 2. Mix a small amount of dry powdered tgg 
albumin with an excess of soda lime. Put the mixture 
in a dry test-tube and heat gently. Test the vapors that 
escape with moist litmus paper. What does this show? 

ExPT. 3. Place about 5 cc. of dilute NaOH in a test- 
tube with a small quantity of tgg albumin and add 



Laboratory Manual 15 

two or three drops of lead acetate. Boil the mixture a 
few minutes and note the change. What is the dark- 
colored precipitate ? 

ExPT. 4. Mix some of the dry egg albumin with 
double its quantity of fusion mixture (Na2C03 4- 
KNO3). Place in a crucible and heat cautiously until 
the mixture becomes colorless. Dissolve the residue in 
warm water. Acidify one portion of the solution with 
HCl and add BaClg. What is the white precipitate? 
Write the equation. To another portion add HNO3 ^^^ 
ammonium molybdate solution, warm to 35° C./ and 
shake. What is the yellow precipitate? 



GENERAL REACTIONS OF PROTEINS 

Much has been learned concerning the constitution 
of protein substances by a study of the decomposition 
products. This decomposition may be accomplished by 
oxidation or by hydrolysis. The products thus formed 
exhibit a great variety of properties due to the presence 
of different radicals or groups of radicals, so no one 
reaction is distinctive for all proteins. 

Among the most important reactions used for identi- 
fication are those which give a distinctive color and those 
obtained by precipitation. 

Prepare a 2 per cent solution of white of egg as 
follows : Put the white of an egg in an evaporating dish, 
cut with the scissors, and then dilute 20 cc. to i liter with 

^ The Centigrade scale is used in all these experiments. 



1 6 Food and Nutrition 

distilled water. Shake thoroughly and filter. Reserve 
the solution for the following tests : 

Color Reactions 

ExPT. 5. Biuret reaction. — To 5 cc. of the albumin 
solution add an equal value of NaOH or KOH. Add 
slowly dilute CuSO^ solution. Notice color. 

ExPT. 6. Xanthoproteic reaction. — To 5 cc. of the 
solution add an equal volume of concentrated HNO3. 
Heat until you obtain a yellow precipitate or a yellow 
solution. Cool and add an excess of NH4OH. Note 
change of color. 

ExpT. 7. Millon's reaction. — To 5 cc. of the solution 
add a few drops of Millon's reagent. Heat cautiously. 

ExPT. 8. Adamkiewicz's reaction. — To 2 cc. of con- 
centrated H2SO4 add about 4 cc. (two volumes) of 
glacial acetic acid ^ and shake. To the mixture add 
one drop of the undiluted G.gg albumin. The liquid 
changes, slowly on standing, more rapidly when slightly 
warmed, to a beautiful reddish violet color. This re- 
action is not given by gelatin. Why? To what are the 
preceding reactions due? 

Report the results obtained with albumin, globulin, 
proteose, peptone, and gelatin, in tabular form, with 
the reagents you have used. 

1 This color reaction is due to the presence of glyoxylic acid in the 
acetic acid. The Hopkins-Cole test modifies the above by using a 
glyoxylic acid solution in place of the acetic acid prepared as follows : 
One liter of a saturated solution of oxalic acid is reduced by 60 grams 
of sodium amalgam and allowed to stand until the evolution of a gas 
ceases, filtered and diluted with two to three volumes of water. 



Laboratory Manual 



i; 





Albumin. 


Globulin. 


Proteose. 


Peptone. 


Gelatin. 


Water 












Alcohol 












Ether 












Biuret 












Xanthoproteic .... 












Millon's 












Heat 












HgCl2 












Pb(C2Hs02)2 












AgNOs 












HNO3 












H0SO4 












HCl 












H(C2H302) strong . . . 












HC2H3O2 + NaCl . . . 












HC2H3O2 + KiFe (Cn)6 












Picric acid 












Tannin 












(NH4)2S04 (saturated) . 












MgS04 (j^ saturated) . 












NaCl (saturated) . . . 












Dilute salt solution . . 

























1 8 Food and Nutrition 

Precipitation 

ExPT. 9. To about 3 cc. of white of egg solution 
add excess of strong alcohol. Does this produce a pre- 
cipitate? Does alcohol coagulate proteid? 

ExPT. 10. Dilute 5 cc. of the albumin solution with 
twice its bulk of o.i per cent H2SO4. Add ether and 
shake briskly. Note the result. 

ExPT. II. To 3 cc. of albumin solution add a drop 
or two of mercuric chloride. A white precipitate forms. 
Repeat experiments with lead acetate and silver nitrate. 

Why is white of egg given in cases of poisoning with 
metallic salts? 

ExPT. 12. Try the effect of the following reagents 
on egg albumin, using 3-5 cc. of the albumin solution : 

HCl, HC2H3O2 strong, 

H2SO4, HC2H3O2 strong -|- excess NaCl, 

HNO3. 

ExPT. 13. To about 5 cc. of albumin solution add 
one to two drops strong acetic acid and then one to two 
drops of potassium ferrocyanide. Does this give a 
precipitate ? 

ExPT. 14. Place 20 cc. of 5 per cent egg albumin 
solution in a beaker. Add powdered (NH4)2S04 to 
saturation, and keep in a water-bath at about 35° for 
half an hour. Stir frequently until the salt ceases to 
dissolve. Note the formation of a precipitate. Filter 
and test the filtrate with the biuret test and the precip- 
itate with Millon's test. Explain the results. When 



Laboratory Manual 19 

making the biuret test in the presence of (NH4)2S04 
or MgSO^ add a large excess of KOH in soHd form. 

ExPT. 15. Place 20 cc. of 5 per cent Qgg albumin 
solution in a beaker and add powdered MgSO^ to satura- 
tion; digest with frequent stirring at 35° for about 
half an hour. Observe formation of a precipitate. Filter 
and test the filtrate and precipitate as in the above ex- 
periment, using the same caution in making the biuret 
test What protein is present in the filtrate? 

Coagulation by Heat 

ExPT. 16. Place about 5 cc. undiluted tgg albumin 
in a test-tube with a perforated cork, through which 
passes a thermometer. The bulb of the thermometer 
should be immersed in the albumin. Suspend the tube 
in a beaker of water and heat gradually. Note the tem- 
perature at which the albumin clouds. Note the temper- 
ature at which it becomes solid. The albumin is heated 
more evenly if the water is stirred during heating. Try 
Millon's and Xanthoproteic tests on the coagulated 
protein. 

Heat and Reagents 

ExPT. 17. In each of four test-tubes place 5 cc. of 
the e.gg albumin solution (1-50). To tubes i and 2 
add respectively i cc. and ^ cc. of a 10 per cent NaCl 
solution. To the tubes 3 and 4 add respectively one 
and five drops of a i per cent acetic acid solution ( i cc. 
of glacial acetic diluted to 100 cc). To a fifth tube 
containing 5 cc. of egg albumin solution ( i-io) add i cc. 



20 Food and Nutrition 

of lo per cent NaCl solution. Immerse the five tubes 
in a bath of boihng water for about five minutes, then 
examine and note the results. Now add one or two 
drops of the i per cent acetic acid to tubes i, 2, 5, and 
to tube 4 add i cc. of 10 per cent NaCl, and heat again. 
What is the result? 

Globulin 

See Meat 

See Flour 
ExPT. 18. Extract 20 grams of crushed hemp seed 
with 100 cc. of 5 per cent solution of NaCl for one-half 
hour at 60° C. Filter while hot through a paper moist- 
ened with 5 per cent NaCl solution, and allow the filtrate 
to cool slowly. Upon cooling, the globulin separates out 
in crystalline form. 

(a) Try two color reactions and two precipitation 
tests on portions of the filtrate. 

(b) Test the coagulability of the filtrate. 

(c) Pour some of the solution drop by drop into a 
beaker of water. 

Phospho-Proteins ( Nucleo-Albumin ) 
For separation of casein from milk see pages 45, 

46, 47- 

ExPT. 19. (a) Apply the Millon and the Xanthopro- 
teic tests to powdered casein. 

(b) Try its solubility in water and in a weak NaaCOg 
solution. 

(c) Test some of the casein for phosphorus as 
follows : 



Laboratory Manual 21 

Grind about 0.2 gram of the casein with four times 
its volume of a mixture made up of equal parts of 
NagCOg and KNO3, and fuse until the evolution of gas 
has ceased. Cool, dissolve in a small amount of warm 
water, acidify with HNO3, and add 5 cc. of ammonium 
molybdate solution and shake vigorously for five minutes. 
Precipitation will take place more rapidly from warm 
solutions, but care must be taken not to heat above 70° C. 
or M0O3 may be precipitated. 



DERIVED PROTEINS 
Albuminates 

ExPT. 20. Acid Albuminate. — Dilute white of tgg 
with about four volumes of water. To 25 cc. of the 
solution add 5 cc. of two-tenths per cent HCl and warm 
on the water-bath for two hours at about 45°. Filter 
and neutralize the filtrate with dilute NaOH solution, 
being careful not to get enough to redissolve the precipi- 
tate. Dissolve some of the precipitate in a little water 
to which dilute HCl has been added. Boil some of this 
solution. Does it coagulate? Add some NaCl. Does 
this cause precipitation ? 

ExPT. 21. Alkali Albuminate. — Put the white of an 
tgg in an evaporating dish and add concentrated KOH, 
drop by drop, with constant stirring. The mass will 
gradually assume the consistency of jelly. An excess of 
KOH dissolves the jelly. Cut the jelly in pieces and 
wash on a cloth with water. Dissolve some of the jelly 
in water (sufficient alkali is present to make a weak 



22 Food and Nutrition 

alkaline solution) by means of gentle heat. Cool and 
neutralize with dilute HCl. Does the alkali albuminate 
precipitate ? 

Proteoses 

Proteoses and peptones may be prepared from com- 
mercial peptones such as Witte's. 

ExPT. 22. Prepare a 20 per cent solution of the pep- 
tone. Heat sufficiently to make the solution complete. 
Put some of this solution in a test-tube and heat to boil- 
ing. Does it coagulate? 

ExPT, 23. To 10 cc. of the solution in an evaporating 
dish add 10 cc. of saturated (NH4)2S04 solution. Does 
a precipitate form? Add about 8 grams of powdered 
(NH4)2S04, and let it stand in a water-bath at about 
25°-32° for one-half hour. Notice the sticky precipitate 
that adheres to the rod and to the sides of the beaker. 
Transfer the precipitate to a filter and wash with about 
10 cc. of saturated (NH4)2S04 solution. 

By means of a glass rod gather up the sticky proteose 
precipitate and transfer it to about 20 cc. of water in a 
test-tube. While stirring, heat the liquid carefully, and 
the proteose dissolves completely. 

With this aqueous solution of proteose make the 
following tests, employing small quantities of the liquid : 

(a) Biuret test. 

(6) Precipitation with HNO3. 

{c) Picric acid. 

{d) Acetic acid and potassium ferrocyanide. 



Laboratory Manual 23 

Peptones 

ExPT. 24. To some of the filtrate from Experiment 23 
add excess of solid KOH and try the biuret test. 

For the preparation of peptone and further tests see 
pages 63, 64, 71, 72. 

COMPOUND PROTEINS 

Glucoproteins 

ExPT. 25. Mucin. — Obtain saliva by chewing some 
paraffin to start the flow of the secretion. Filter the 
saliva and use it in the following experiments. 

ExPT. 26. Add 60 cc. of saliva to 200 cc. of 95 per 
cent alcohol. Filter off the precipitated mucin and make 
the following tests : 

(o) Try some color-reactions for proteins. 

(&) Dissolve some of the precipitate in weak NaOH 
and then add dilute acetic acid, drop by drop. Note 
results. 

(r) Boil the remaining precipitate in 25 cc. of 10 
per cent (by volume) HCl for two to four hours. The 
result is much more satisfactory if a condenser is used 
to prevent evaporation. Cool and render the solution 
alkaline with concentrated KOH ; filter, and then test for 
reducing substance by Fehling's solution. Let the solu- 
tion stand, as the reduction may not be evident at first. 

Nucleo-Proteins 
In recent years much interest has been shown in this 
group of the compound proteins, partly because of their 
relation to the purin bodies. 



24 Food and Nutrition 

Nucleo-proteins consist of protein and nucleic acid 
or nuclein. They are widely distributed in the animal 
body, and while they occur chiefly in cell nuclei they are 
also found in the protoplasm. 

Particular importance attaches to their decomposition 
products. On digestion with pepsin hydrochloric the 
more complex nucleo-proteins are split into protein sub- 
stance and a simpler nucleo-protein rich in phosphorus, 
known as nuclein. On further decomposition this nuclein 
yields nucleic acid, which serves to distinguish this group 
from phospho-proteins or nucleo-albumins. 

On hydrolysis they yield three types of decomposition 
products : 

1. Purin bases — xanthin, guanin, hypoxanthin. 

2. Pyrimidin derivatives — mucin, thymin. 

3. A carbohydrate group. 

As purin bases upon oxidation yield uric acid, the 
importance of nucleo-proteins is readily understood. 

Nucleo-protein may be prepared in the laboratory 
from the pancreas. See page 70. 

ALBUMINOIDS 

Gelatin 

ExPT. 27. Use a 2 per cent solution of commercial 
gelatin, or prepare gelatin from tendon. 

Try the following tests : 

(o) Three color tests. 

{h) Behavior with H2SO4, alcohol, tannic acid, and 
AgN03. 



Laboratory Manual 25 

ExPT. 2^. Procure a tendon, cut it into small pieces, 
and to 10 grams add 200 cc. of distilled water and 3 cc. 
of dilute acetic acid. Boil one-half hour, strain through 
cheese cloth, and evaporate it on the water-bath to 15 cc. 
Transfer it to a warm test-tube, insert a thermometer, 
and note the temperature at which the gelatin solidifies. 

Decomposition of Proteins 

In studying the constitution of protein substances 
they have been separated into simpler compounds. These 
compounds are known as dissociation or decomposition 
products. Through the work of E. Fischer and his 
associates, many of these products have been obtained. 
Mann groups them as follows : 

(a) Open-chain amino-acids ; (/?) ring compounds; 
(c) ammonia; {d) thio-amino acids. Leucin, glycocoll, 
and tyrosin are all members of the first group, and at 
the same time the best known of all the dissociation 
products. Leucin was the first of these products to be 
discovered. 

For the preparation of tyrosin and leucin see Diges- 
tion, page 64. 

Questions 

1. Name the forms of protein that you have obtained 
in the laboratory. 

2. Name some common food in which each may be 
found. 

3. What protein substances would you expect to 
find in the following: raw custard, cooked custard, con- 
somme, beef tea ? 



26 Food and Nutrition 

4. Have you been able to deduce any principles 
concerning the cooking of foods rich in proteins? 

5. Distinguish between precipitation and coagulation. 

6. Give some commercial use of the coagulation of 
proteins. 

7. In what forms may protein be precipitated? 

8. Explain the use of fractional coagulation. 

9. Why is tannin used in curing hides? 

10. Why is white of ^gg given in cases of lead 
poisoning? Explain the use of white of ^gg in the 
clearing of soups. 

11. Is there any reason why vinegar should be used 
in the preparation of tough meat? 

12. How is the setting of gelatin explained? 

13. How is this power lost or destroyed? 

14. What is the chief use of gelatin in the household? 

15. How does it rank among food materials? 



FATS 

Chemically considered the fats are glycerides of the 
fatty acids. They are widely distributed in nature in 
both plants and animals. When used as food they be- 
come an important source of energy for the body, as they 
are rich in carbon. 

References: Hammarsten, Allen, Sherman, Thorpe, Leff- 
mann and Beam. 

ExPT. 29. Compare some of the common fats, such 
as tallow, lard, and butter, with respect to their color, 
odor, and taste. 

ExPT. 30. Place a drop of fat on a piece of paper. 
Do the same with a drop of ethereal oil and compare the 
results upon standing. 

ExpT. 31. Test a small portion of rancid fat by dis- 
solving in alcohol and adding a solution of neutral litmus 
or red rosalic acid. To what is the rancid odor due ? 

ExPT. 32. Test the solubility of fats in water, alcohol, 
ether, and chloroform. 

ExPT. 33. Shake a drop of neutral olive oil with 
about 5 cc. of water in a test-tube. Repeat, using a very 
dilute NagCOg solution, a soap solution, and a dilute 
albumin solution, in place of the water. Compare the 
permanency of the emulsions so formed. What is an 
emulsion ? 

ExPT. 34. Saponification. — Dissolve 15 grams of 
KOH in 10 cc. water, add to the solution 100 cc. of 90 

27 



28 Food and Nutrition 

per cent alcohol, and heat on the water-bath. Add to 
this solution 50 grams of lard which has been dissolved 
by warming in an equal volume of alcohol. Saponifica- 
tion takes place readily on mixing the solutions, and is 
complete when a drop of the solution dissolves completely 
in water, leaving no fat globules suspended. The fatty 
acid may be obtained from this alcoholic soap solution 
by bringing to acid reaction with dilute HoSO^ and cool- 
ing, when the fatty acid comes to the top and solidifies 
as a cake, which may be further purified by melting in 
hot water, and after vigorous stirring cooling to the 
solidification temperature again. 

Save the alcoholic solution for the preparation of 
glycerol. 

Soap from Fatty Acid and NaoCOo 

ExPT. 35. Melt a small amount of the acids prepared 
from lard in a beaker on the water-bath. Add grad- 
ually, with constant stirring, a half-saturated NaoCOg 
solution until all the fatty acid has dissolved. This may 
take some time ; an excess of NaXOa should be avoided. 
After solution has been accomplished pour a small por- 
tion into a test-tube and add an equal volume of cold 
water. A jelly will form. Immerse another portion in 
a dish of cold water. Soap is precipitated from solution. 
Allow the rest to stand and cool slowly. 

Separation of Glycerol 

ExPT. 36. Filter the solution remaining after pre- 
cipitation of the fatty acids and make it nearly neutral 



Laboratory Manual 29 

with NaOH solution, and then neutrahze with NagCOg 
solution. Evaporate nearly to dryness, first over the 
free flame and then over the water-bath. Mix the resi- 
due with about 50 cc. of 90 per cent alcohol. Let this 
solution stand for some time, and then filter and evap- 
orate the filtrate on the water-bath. Dissolve the residue 
in absolute alcohol, so that the mixture makes about 
25 cc. Add 25 cc. ether, shake, and let it stand until the 
next day. Filter and evaporate the filtrate cautiously 
on the water-bath. The glycerol is obtained as a light 
yellow sirup. The acrolein test may be tried as follows : 

Mix a small amount of the glycerol with some 
powdered potassium bisulphate and heat the mixture 
in a dry test-tube. Notice the penetrating odor. Test 
the fumes with some filter paper moistened with some 
ammoniacal silver oxide solution. The paper turns black. 
What does this show ? 

The ammoniacal silver solution is prepared by adding 
to 5 cc. of silver nitrate solution one half the volume of 
NaOH solution and dissolving the precipitation in 
NH.OH. 

ExPT. 37. Melting point of fats. — Find melting 
points of butter, lard, beef fat, and mutton tallow accord- 
ing to the method used in work on food analysis or in 
"Principles and Practice of Agricultural Analysis" 
(Wiley). To what is the difference in melting point 
due? 

Lecithin 

ExPT. 38. Preparation of egg lecithin. — Separate 
the yolks of two eggs from the white. Mix the yolks 



30 Food and Nutrition 

with 60 cc. of ether and let stand over night ; in the 
morning add 100 cc. of alcohol and filter the solution. 
Evaporate the filtrate on the water-bath. Dissolve the 
residue from the filtrate in 15 cc. of ether; filter, and 
add 50 cc. of acetone to the filtrate obtained. Filter this 
through a small filter and save the filtrate for cholesterin. 
With the lecithin thus obtained perform the following 
experiments : 

(a) Add a small piece of lecithin to water; shake; 
notice cloudy appearance of water. No true solution 
takes place, but an emulsion is formed which can be 
filtered unchanged. Notice myelin forms, under micro- 
scope. 

{h) Tests for phosphorus. — Fuse one-fourth of the 
lecithin with a fusion mixture in a porcelain crucible. 
When well fused allow to cool, dissolve in water, acidify 
with nitric acid, and add ammonium molybdate. Heat 
to 50° and shake. Does a yellow precipitate form? 

(c) Tests for fatty acid. — Heat one-third of the 
lecithin with 20 cc. of sodium alcoholate in a flask for 
one hour; evaporate the alcohol. Notice the residue. 
It is soap. Dissolve this in water; boil, and notice the 
formation of soap bubbles. Make the solution acid and 
allow it to stand in a warm closet over night. Fatty 
acids collect at the surface. Cool on ice and the layer 
of fatty acids may be removed. 

{d) Tests for glycerin. — Fuse a small portion of 
the lecithin with some potassium bisulphate. Note the 
peculiar irritating odor of acrolein given off. This shows 
presence of glycerin. Give the formula for lecithin. 



Laboratory Manual 31 

(e) Test for nitrogen. — Fuse some lecithin in a 
test-tube with a piece of metalHc sodium or soda Hme 
the size of a pea. Notice the smell of ammonia and test 
with a moist piece of red litmus paper. 

Cholesterin 

ExPT. 39. Preparation of cholesterin. — Evaporate 
the acetone solution from which the lecithin has been 
precipitated on the steam-bath. Dissolve the residue 
in 50 cc. of alcoholic sodium hydroxide (10 per cent) 
and heat on the steam-bath in a flask until all the alcohol 
has evaporated. Dissolve the residue in 50 cc. of water, 
transfer to an evaporating dish, add 10 grams of sodium 
chloride, and again evaporate to complete dryness. Grind 
this residue fine in a mortar, dry at 105°, and extract 
with 30 cc. of cold ether. Filter the ether solution, and 
crystals of cholesterin remain after evaporation of the 
ether. 

Reactions of cholesterin: 

(a) Evaporate with nitric acid. A yellow mass is 
obtained. Add ammonia. What change takes place? 

(b) Mix in the dry state with strong sulphuric acid. 
Add chloroform. Note change. Does it change further 
on exposure to air ? 

(c) Evaporate with a mixture of two volumes of 
sulphuric acid and one volume of ferric chloride solution. 
Is there a change of color? 

(d) Dissolve in chloroform and observe crystals 
under microscope. Write the formula for cholesterin. 



32 Food and Nutrition 

Questions 

1. To what series of fatty acids are the fats related? 

2. Give an example of an unsaturated fatty acid and 
a saturated fatty acid. 

3. How do you distinguish oils, fats, and waxes ? 

4. Why is linseed oil used in paint? 

5. What forms of fat have you worked with in your 
laboratory courses? 

6. Name some of the ethereal oils that you have 
used in the laboratory. 

7. Do they all come under one classification in 
chemistry ? 

8. How is renovated butter made? oleomargarine? 

9. What change takes place when fats become 
rancid ? 

10. What is the effect of heat on fats? 

11. What makes the fumes of hot fat irritating? 

12. What is the effect of cold on fats? 

13. What are the qualities to be desired in a fat used 
for cooking purposes ? 

14. What are the relative merits of beef suet, mutton 
tallow, lard, and olive oil for cooking purposes ? 



CARBOHYDRATES 

This is a name applied to a class of compounds which 
are especially abundant in the vegetable kingdom. While 
protein bodies form the larger part of the solids in 
animal tissue, carbohydrates form the chief part of the 
plants, and occur in the animal kingdom only in small 
quantities. Owing to their wide distribution, ease of 
digestion, and relative cheapness they constitute a very 
important source of food. Because of their easy oxida- 
tion they are one of the principal sources of energy in 
the animal body. Carbohydrates are compounds of 
carbon, hydrogen, and oxygen. The statement frequently 
made that the carbohydrate molecule contains six atoms 
of carbon or a multiple of six, and that the H and O 
are in the proportion to form water is not true in all 
cases. Example, Rhamnose CeH^oOg. 
References: Hammarsten, Richter-Smith, W. A. Noyes. 

The principal divisions of the carbohydrates are: 
I. Monosaccharides or Glucoses, CgHisOg. 
II. Disaccharides or Sucroses, QaHgoOn. 

III. Polysaccharides or Amyloses, (C6Hio05)n. 

Monosaccharides are further divided (see table), 
according to the number of carbon atoms they contain, 
into trioses, tetroses, pentoses, hexoses, etc. Again, they 
are classified according to derivation into aldehydes and 
ketones. Ordinary glucose is an aldehyde; ordinary 
fructose is a ketone. By oxidation (see table) the alde- 
hydes yield acids ; ordinary glucose yields gluconic acid, 
and, on further oxidation, saccharic acid. 

33 



34 



Food and Nutrition 



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Laboratory Manual 
DIVISIONS OF CARBOHYDRATES 



35 





Example. 


Preparation and occurrence. 


Monosaccharides 






Trioses CsHeOg 


Glycerose 


Oxidation of glycerin 


Tetroses C4H8O4 


Erythrose 


Oxidation of erythrite which 
is found in some plants 


Pentoses C5H10O5 


Arabinose 


Action of acid on cherry gum 




Xylose 


Action of acid on wood gum 




Rhamnose 1 


Decomposition of various 
glucosides 




Fucose 


Action of acid on sea weed 


Hexoses CeHigOe 


Mannose 


Oxidation of mannite which is 
found in plants 




Dextrose 


Widely distributed in the veg- 
etable kingdom, as in sweet 
fruits, honey, etc. 




Galactose 


Action of dilute acid on milk 
sugar 




Levulose 


Found in connection with dex- 
trose in vegetable kingdom 




Sorbinose 


Found in the juice of service 
berries 


Heptose C7H14O7 






Octose CsHieOg 






Nonose CgHigOg 






Disaccharides C12H22O11 


Sucrose 


Widely distributed in nature 




Maltose 


Found in germinating cereals 




Lactose 


Found in the milk of animals 


Trisaccharides C18H32O16 


Raffinose 


In sugar beet 


Polysaccharides (C6Hio05)n 


Pentosans 


Found in plants, wheat bran 




Cellulose 


In nature as cell wall of plants 




Starch 


Found in plants as reserve 
material 




Dextrin 


By heating starch to 200-210° 




Glycogen 


Found in the liver 




Inulin 


Found in the roots of com- 
positae 




Lichinin 


Found in lichens 




Pectin 


Found in fruits and vege- 
tables 



CeHiaO.; CH-AHoOs 



36 Food and Nutrition 

The carbohydrates which have the greatest physio- 
logical importance and are most used for food are either 
hexoses or pentoses. A list of the more common forms, 
with the place of their occurrence, is in the foregoing 
table. Later, typical forms of the different classes will 
be studied. 

ExPT. 40. Compare dextrose, levulose, cane sugar, 
lactose, dextrin, and starch as follows : 

1. Solubility in cold water. 

2. Trommer's test and Fehling's test. 

3. Iodine. 

4. Molisch's test. 

5. Fermentation. 

(a) Solubility. — Take 10 cc. water, add 4 grams of 
the substance to be examined, and shake thoroughly. 
When the substance is dissolved add 4 grams more, and 
so on until no more dissolves. 

Dilute the solutions already made to 2 per cent and 
use them in the following experiments. 

(b) Trommer's test. — To 5 cc. of the solution add 
about one-half its volume of KOH solution. Shake and 
add, drop by drop, a dilute solution of cupric sulphate. 
When a slight permanent precipitate of cupric hydroxide 
appears, heat and notice if reduction takes place. 

(c) Fehling's test. — Put a small amount of Fehling's 
solution in a test-tube and add four times its volume of 
water. Boil, to see that the solution itself does not cause 
precipitate of cuprous oxide. If the precipitate forms, 
a new solution must be prepared. Add the solution to 
be tested a few drops at a time and boil after each addi- 



Laboratory Manual 37 

tion. A yellowish or a brownish red precipitate shows 
that reduction has taken place. 

(d) Iodine. — To 5 cc. of each solution add a drop 
or two of iodine dissolved in potassium iodide. Note 
color. 

(e) Molisch's test {Hawk). — "Place approximately 
5 cc. of concentrated H2SO4 in a test-tube. Incline the 
tube, and slowly pour down the inner side of it approxi- 
mately 5 cc. of the sugar solution to which 2 drops of 
o-naphthol solution (about 15 per cent alcohoHc solution) 
has been added, so that the sugar solution will not mix 
with the acid. A reddish violet zone is produced at the 
point of contact." 

(/) Fermentation. — Mash up a compressed yeast 
cake in 16 cc. of water ; place i cc. of this homogeneous 
mixture in each of six fermentation tubes, then add to 
each fermentation tube 10 cc. of a 5 per cent solution 
of each of the substances to be tested. Examine after 
ij^ hours and after 24 hours. 

Name the products of fermentation obtained in the 
above experiment. 

Glucose and Levulose 

Use a 2 per cent solution of glucose unless otherwise 
indicated. 

ExPT. 41. Place some of the dry glucose in a tube 
and heat gently over a flame. Note changes. The 
peculiar odor is that of burnt sugar. Allow the tube to 
cool, then add water and warm slightly. Explain results. 

ExPT, 42. To some dry glucose add cold, concen- 



38 Food and Nutrition 

trated H2SO4 and let stand. The liquid remains color- 
less, or at most is light yellow. Compare with similar 
experiment under cane sugar. Then gently heat the 
glucose tube. What is formed? 

ExpT. 43. To some 95 per cent alcohol in a test-tube 
add a few cc. of dextrose solution. Compare with ex- 
periment under dextrin. 

ExPT. 44. Formation of osazones from dextrose. — 
In a test-tube prepare a mixture of 5 drops of phenyl- 
hydrazin, 10 drops of glacial acetic acid, and i cc. of 
saturated salt solution, and boil for a few minutes. 
Yellow phenylglucosazone crystals will appear on cool- 
ing. Write the equations for the formation of the osa- 
zones. For more exact experiments see Sherman. 

ExPT. 45. Try the effect of dry heat and H2SO4 on 
cane sugar (see experiments under dextrose). 

ExPT. 46. Place 50 cc. of the cane sugar solution in 
a small beaker, add 6-8 drops of concentrated HCl, and 
boil for 2-3 minutes. Has the cane sugar been inverted? 

ExPT. 47. Compare the sweetening power of cane 
sugar, dextrose, and commercial glucose. 

Lactose 
For preparation of lactose see milk. 

Maltose 

For formation of osazone see dextrose. 

ExPT. 48. Hydrolysis of starch by maltose. — Make 
a malt extract by allowing 5 grams of malt to soak in 
50 cc. of water for i>^ to 2 hours at room temperature. 



Laboratory Manual 39 

This malt extract contains some reducing substances, 
so in order to prove the formation of a reducing sub- 
stance by the action of malt on starch it is necessary to 
proceed as follows : 

Add to one Erlenmeyer flask 50 cc. of a 5 per cent 
solution of starch paste and about i cc. of chloroform and 
mark A. To another Erlenmeyer flask add 50 cc. of 
water and i cc. of chloroform and mark B. Then to 
each add 10 cc. of the malt extract, cork, and allow to 
stand until the starch solution gives no color reaction 
with iodine. This will take two or three days. Prepare 
two test-tubes containing the same amounts of Fehling's 
solution (about 5 cc. after dilution) and heat to boiling. 
To the first add 10 drops of the pure malt solution 
(flask B) from a pipette, and to the second the same 
amount of the reaction mixture of malt on starch 
(flask A). Compare the reducing powers of the two 
solutions. 



POLYSACCHARIDES 

Starch 

ExPT. 49. Examine wheat, potato, oat, and corn 
starch under the microscope. Make drawings. 

ExPT. 50. Soak dried peas in water over night, then 
cut thin slices and examine under the microscope. 

ExPT. 51. Examine with the microscope raw, boiled, 
baked, and mashed potato. Cut thin slices when possi- 
ble. Make drawings to represent the difference in 
appearance. 



40 Food and Nutrition 

ExPT. 52. Place 100 cc. of water in a beaker and boil 
it; then add i gram of powdered starch and continue 
boiling for 2-3 minutes, stirring constantly. Reserve 
for following experiment. 

ExpT. 53. To about 50 cc. of the starch solution in 
a beaker add ^ cc. of H2SO4, cover with a watch-glass, 
and boil for 15 minutes. Replace the water that may 
be lost by evaporation. Now place some of the liquid 
in a tube; render alkaline with sodium or potassium 
hydroxide. Make Fehling's test. If no reduction takes 
place continue heating the contents of the beaker for 
another 15 minutes and test as before. Has inversion 
taken place? 

ExPT. 54. Spread one teaspoonful of wheat starch 
on a tin plate and heat in the oven in which a ther- 
mometer is suspended. When the oven temperature 
reaches 200° take out a small amount of starch and test 
with iodine on a white plate. Repeat the test for each 
rise of 10°. How are these changes explained? 

ExPT. 55. Gelatinization of starch. — Put i gram 
of potato starch into 100 cc. of water. Shake well, then 
distribute this solution equally among ten test-tubes. 
Put a rubber band around these tubes and place them 
in a water-bath. Heat gradually to 55° C. Then remove 
one tube and cool it quickly. Repeat for each rise of 5°. 
Boil the last tube over the free flame. Place in test-tube 
rack, let stand, and compare. Look at a drop from each 
test-tube under the microscope and make drawings. 

This experiment may also be tried using 3 grams of 
potato starch and 100 cc. water. In this case there is 



Laboratory Manual 41 

sufficient starch present to form a paste when it is heated 
to the proper temperature. 

ExPT. 56. Test for starch, substances in which it is 
Hkely to be found as an adulterant or extender. 

Dextrin 

ExPT. 57. Examine dextrin under the microscope. 

ExpT. 58. Add tannin to a solution of dextrin. Is 
a precipitate formed? 

ExPT. 59. Add alcohol to a solution of dextrin. 
What happens ? 

ExPT. 60. Obtain dextrin from toasted bread. Is 
toasted bread thoroughly dextrinized? 

ExPT. 61. Chop a half pint of oysters as fine as 
possible. To this material add 500 cc. of boiling water 
slightly acidulated with acetic acid. Strain the liquid 
through muslin. This liquid contains, besides glycogen, 
some proteins and gelatin. To remove the latter, first 
concentrate to a small volume, then add alternately a 
few drops of HCl and of potassium mercuric iodide till 
a precipitate ceases to form. Finally, filter off a little of 
the liquid and test it with acid and reagent to make 
sure that all the proteins are precipitated. If this is the 
case, strain the liquid through muslin, then filter through 
paper, and to the filtrate add two volumes of alcohol and 
stir thoroughly. Allow the glycogen to settle, then filter 
off, wash with dilute alcohol (2 parts alcohol to i part 
water). Finally transfer to a beaker, cover with abso- 
lute alcohol, and let stand an hour or more. Then filter 
off the glycogen, fold the filter, and gently squeeze off 



42 Food and Nutrition 

excess of alcohol ; finally press between several layers of 
filter paper till dry. Powder, and save for use later. 

(o) To some glycogen in a small beaker add 20-30 cc. 
of water and warm. The glycogen dissolves, forming 
an opalescent liquid. 

{h) To a portion of the solution just obtained add 
a few drops of iodine solution (in potassium iodide). 
A reddish brown color forms. Then heat the contents of 
the tube. The color disappears, to reappear on cooling. 

{c) Boil another portion of the glycogen solution 
with Fehling's solution. Note the result. 

{d) To some of the glycogen solution add a few 
drops of HCl and boil a few minutes. Then cool and 
neutralize, and test a portion with iodine. Try Fehling's 
on another portion. 

Cellulose 

Hydrolysis of Cellulose 
ExPT. 62. To one sheet filter paper add 15 cc. H2SO4 
(50 per cent). To this add gradually enough concen- 
trated H2SO4 to bring the filter paper into solution. If 
this H2SO4 solution is tested with iodine directly, or upon 
allowing a few drops of water to run onto it, but not 
get thoroughly mixed with it, a blue starch iodide color 
results. If the amylocellulose solution is precipitated by 
dilution with water (three times volume), then iodine 
added, no color is given. Dilute to about 30 cc. with 
water and boil one hour. Make the solution alkaline 
with potassium hydroxide and test with Fehling's 
solution. 



Laboratory Manual 43 

Pectin 

ExPT. 62,. Preparation of pectase. — Pare young 
carrots and reduce them to a pulp. Express the juice, 
filter, and save it to add to the solution of pectin. 

ExPT. 64. Preparation of pectin. — Put the residue 
from the previous experiment into a beaker, cover with 
water, and boil 15 minutes. Filter. Put 5 cc. of the 
solution into a test-tube and add a few drops of the liquid 
containing the pectase. Does the juice form a jelly? 
How long does it take to form a jelly? Extract pectin 
from apples and repeat the above experiment. Does acid 
interfere with the action of pectase ? What is the reason 
for the non-production of jelly in the juice of many 
fruits ? 

Questions 

1. What are the legitimate uses of commercial 
glucose in the home ? 

2. In what form are the carbohydrates of food 
absorbed in the body? 

3. Where and in what forms do glucose and levulose 
occur ? 

4. When cane sugar, lactose, and maltose are hydro- 
lyzed, what monosaccharides are obtained? 

5. What changes take place in boiling sugar for 
fondant ? 

6. What factors influence the final temperature in 
making fondant? 

7. Why is acid or glucose added in making candy? 

8. What is the source of commercial lactose ? What 
is its use? 



44 Food and Nutrition 

9. What is the commercial use of maltose? 

10. How is dextrin prepared commercially? 

11. Do our cooking processes affect cellulose? 

12. Where is pectin found? 

13. What transformations does pectose undergo in 
fruits ? 

14. What transformations does pectin undergo in 
jelly making? 

15. Discuss the problems of jelly making. 

16. What principles of starch cookery are you able 
to deduce from above experiments? 

17. How would you separate fructose and glucose 
from invert sugar? 

18. How does the strength of the acid influence the 
rate of inversion of cane sugar ? 

19. Compare HCl and HC2H3O2 in this respect. 



MILK 

References: Salkowski. 

Blyth (5th ed.). 

Sherman. 

Richards and Woodman. 

Leffmann and Beam. 

Conn. Yeasts, Molds, and Bacteria in the 

Home. 
Farmers' Bulletin, No. 74. 
Separation into constituents; use of preservatives. 
ExPT. 65. Examine a drop of milk under the micro- 
scope. Sketch the different sized globules present and 
measure their diameter. 

ExPT. 66. Examine microscopically a drop of skimmed 
milk. What difference is observed between this and 
whole milk? 

ExPT. 67. Boil about 25 cc. of milk in a small beaker 
for 5 minutes. No coagulation proper, but scum may 
form. Remove the scum with a spoon or spatula and 
heat again ; a new scum forms. This formation of scum 
will repeatedly take place. What is the nature of this 
scum? 

ExPT. 68. To about 10 cc. of milk in a test-tube add 
one drop of dilute acetic acid (i-io), then boil. The 
casein is coagulated and carries down with it the fat. 
The serum is clear. 

45 



46 Food and Nutrition 

ExPT. 69. Place 10 cc. of milk in each of five test- 
tubes. To No. I add >4 cc. of very dilute HCl (10 drops 
of HCl to 50 cc. of water). To No. 2 add )4 cc. of 
2 per cent NaaCOs solution. To No. 3 add Y^ cc. of 
saturated (NH^) 2C2O4 solution ( 1-20) . Then add to each 
of these three tubes, and also to Nos. 4 and 5, 2 drops of 
rennet solution, and mix. Heat the contents of tube 
No. 5 to boiling. Then place all the tubes in a water- 
bath at 40° and examine every 3-5 minutes and explain 
results. What can you say of the action of alkali on 
rennet ? of acid ? 

What is the composition of coagulum? What is the 
clear liquid that separates from the coagulum on stand- 
ing? Why does not tube No. 3 coagulate? Continue 
heating tube No. 3 at 40° for about one-half hour, then 
add 2-3 drops of CaClg solution. The liquid instantly 
solidifies. Why ? 

ExPT. 70. To some milk in a test-tube add 1-2 vol- 
umes of ether, close, and shake thoroughly. Do the fat 
globules dissolve? Now add a few drops of NaOH and 
shake again. Observe and explain results. This re- 
action was taken at one time to indicate that the globules 
were surrounded by an albuminous envelope. 

ExPT. 71. To some milk in a test-tube add a few 
drops of NaOH and heat. Result? 

ExPT. y2. To about 10 cc. of milk in a test-tube add 
5 grams of powdered MgSO^ and shake thoroughly. 
Then pour onto a filter, resting in a test-tube, and set 
aside to filter over night. Boil the clear filtrate. Result? 
The casein is precipitated almost completely by MgSO^. 



Laboratory Manual 47 

ExPT. 73. To 5 cc. of milk add 4 volumes (20 cc.) 
of strong alcohol; shake thoroughly and set aside. All 
the proteins present are precipitated. 

ExPT. 74. Add 50 cc. of milk to about 125 cc. of 
water, mix well, and while stirring add dilute acetic acid 
(i-io), drop by drop, till the precipitate becomes coarsely 
flocculent and ceases to increase. Stir thoroughly and 
set aside over night. Do not add more acid than is 
necessary. What does this precipitate contain? Filter 
off the precipitate and allow to drain well, then fold 
over half the filter in the funnel and apply gentle pressure 
with the fingers until no more water can be squeezed out. 

Transfer the precipitate to a small, dry beaker, add 
about 30 cc. of strong alcohol, and stir thoroughly so as 
to dehydrate the caseinogen. Then filter and again 
squeeze the contents of the filter as dry as possible. 
Transfer the precipitate to a small, dry beaker ; add about 
50 cc. of ether and stand over night. Filter, wash with 
ether, and squeeze as dry as possible. 

Spread open the filter on the table ; allow the remain- 
ing ether to evaporate, then powdfer. The white, chalky 
powder is caseinogen. 

(a) The ether filtrate received in a small beaker or 
evaporating dish and evaporated cautiously on the water- 
bath gives the milk-fat. 

(b) The aqueous filtrate from the casein and fat 
precipitate contains albumin and milk sugar. Place it 
in a beaker and boil for 15 minutes, in order to coagu- 
late the albumin. Filter, and treat the filtrate as follows : 
Heat on a wire gauze over flame until it becomes cloudy 



48 ' Food and Nutrition 

and bumps. If the liquid is cooled the cloudiness dis- 
appears. Why? Heat again to boiling and filter hot. 
Concentrate the filtrate on the water-bath, and if the 
cloudiness continues to appear filter several times more. 
When the filtrate is reduced to a sirupy consistency, 
set it aside until the next day. Crystals of milk sugar 
should separate on standing. 

Preservatives 

References: Leffmann and Beam. 
Leach. 
ExPT. 75. Test milk for (a) formaldehyde, {b) sali- 
cylic acid, {c) boric acid, {d) NaaCOg or NaHCOg. 

Questions 

1. What is the problem for the housewife in the 
use of soda and sour milk? 

2. Explain the curdling of milk soups. 

3. What are the sources of danger in ice cream? 

4. Explain the Babcock test used in analysis of 
milk. 

5. What is the use of sucrate of lime in the dairy 
industry ? 

6. Discuss alkaline fermentation of milk. 



MEAT 

References: Mitchell, C. A, Chapters I, VIII, IX, X. 
Grindley and Sprague. A Precise Method 

of Roasting Beef. The University of 

Illinois Studies, Vol. II, No. 4. 
Grindley, H. S. Journal of Amer. Chem. 

Soc, Vol. XXVI, No. 9, September, 1904. 

(Nitrogenous constituents.) 
Grindley and Emmett. Journal of Amer. 

Chem. Soc, Vol. XXVII, No. 6, June, 

1905 ; Vol. XXVIII, No. I, January, 1906. 

(Analysis.) 
Emmett and Grindley. The Chemistry of 

Flesh. 
Trowbridge and Grindley. Chemistry of 

Flesh. Journal of Amer. Chem. Soc, 

April, 1906. 
Grindley and Associates. Bulletins Nos. 102, 

141, 162, 193, U. S. Dept. of Agriculture, 

Office of Experiment Stations. (Cooking 

and digestion.) 
Mann, Gustav. Chemistry of the Proteids. 
Bevier and Van Meter. Selection and 

Preparation of Food. 
ExPT. 76. Put 25 grams of finely chopped meat in 
75 cc of water and allow it to stand for one or two 
hours. Stir frequently. Strain through cheese cloth, 

49 



50 Food and Nutrition 

then filter through paper. Reserve the residue for the 
next experiment. Test the reaction of the solution with 
litmus paper. 

Slowly heat a portion of the filtrate in a test- 
tube in which a thermometer is placed. Put the test-tube 
in a beaker half filled with water and heat the beaker 
on the wire gauze. By frequent stirring of the water 
with a glass rod, on the under end of which is a piece 
of rubber tubing, the even distribution of the heat may 
be accomplished. Even at a slight elevation of temper- 
ature, usually at 45°-56°, coagulation occurs. Filter. 
Heat the filtrate again to 65° and observe results. 
Filter, Heat to 75° and filter. Make two color tests 
for protein with the filtrates. Boil some of the original 
filtrate, filter, and divide into three portions. 

Test one portion with Millon's reagent; to another 
portion add i cc. of a 5 per cent NaCl solution and 5 cc. 
of a 20 per cent tannic acid solution, drop by drop. 
Observe precipitation. To another portion add a few 
crystals of NH4NO3 and 10 cc. of a neutral ammonium 
molybdate solution. Heat to 60° C. This should pre- 
cipitate the inorganic phosphates.^ 

ExPT. yy. Digest the meat residue from the previous 
experiment in 100 cc. of a 15 per cent NH^Cl solution 
for 24 hours in a covered beaker, then filter and test the 
filtrate as follows: 

(a) Pour a part of the solution into a test-tube two- 
thirds full of water. Myosin forms as a cloudlike 
precipitate. 

iThe molybdate solution should have for every 50 cc, of the 
reagent 4 cc. of HN03(i.2 sp. gr.). 



Laboratory Manual 5 1 

{h) Heat some of the solution in a test-tube. Does 
it coagulate? 

{c) Try two color tests. How does this protein 
differ from the one obtained in the first experiment? 
Give the names of the proteins obtained from meat. 

ExPT. 78. Influence of cooking upon the composition 
of meat. 

(a) Boil meat under known conditions. 

1. Examine cooked meat. Repeat experiments 
under raw meat. 

2. Examine broths. Concentrate some of the broth 
and make protein tests. Take 200 cc. of the original 
broth, add 15 cc. of a 10 per cent HCl solution. Evap- 
orate to 25 cc, filter, test for creatinin by Jaffe's reaction. 

(&) Roast meat under known conditions. 

1. Examine roasted meat. Repeat experiments 
under raw meat. 

2. Examine drippings for proteins. 

Extractives 

References: Grindley, H. S., and Woods, H. S. Cre- 
atinin. Jour. Biol. Chem., Vol. H, p. 309. 
Emmett, A. D., and Grindley, H. S. Cre- 
atinin. Jour. Biol. Chem., Vol. HI, p. 491. 
Hammarsten. 
Salkowski. 
ExPT. 79. Creatin and creatinin. — Extract 500 
grams chopped beef with 500 cc. water for half an 
hour over the water-bath at 50°. Strain as dry as 
possible through muslin and make a second extraction 



52 Food and Nutrition 

in the same way, with an equal amount of water (save 
residue). Unite the two extracts, and after concentra- 
tion to about 200 cc. (test 20 cc. for creatinin by Jaffe's 
reaction; also test Liebig's extract in the same manner) 
acidify the solution with 2 or 3 drops of acetic acid. 
Remove the coagulated proteins by filtration, and to 
the filtrate (this filtrate corresponds to Liebig's extract, 
and the extract may be used for study instead of the 
chopped meat) add basic lead acetate, carefully avoiding 
any excess; the precipitate consists of phosphates, chlo- 
rides, sulphates, etc. Allow this to settle and then filter. 
Warm the filtrate and pass HoS through it to remove 
the excess of lead. Filter hot. Concentrate the clear 
filtrate on a water-bath to a thin sirup and allow to 
stand in a cool place. Crystals of creatin will deposit. 
Filter off the crystals, and wash them with 88 per cent 
alcohol. 

Place some of the crystals in a small flask with 
10 cc. dilute H2SO4, and heat for half an hour on the 
water-bath, keeping the volume constant. While still 
warm add powdered BaCOg to neutralization. Filter, 
and evaporate the filtrate to 10 cc. The creatin has been 
changed to creatinin. Write the equation. Perform the 
following tests with the solution: 

(a) Place 2 drops of the solution upon a watch-glass 
and add to it a few drops of an alcoholic solution of 
ZnCl, ; allow it to stand and then examine the crystals 
under the microscope. 

{h) WeyVs reaction. — To 2 cc. of the creatinin 
solution add 3 drops of a freshly prepared dilute solu- 



Laboratory Manual 53 

tion of sodium nitroprusside. Then add, drop by drop, 
dilute NaOH. A ruby-red color is produced, which 
quickly changes to yellow. If the solution is now acid- 
ified with acetic acid and heated, a green color is 
obtained, and upon continued boiling a precipitate of 
Prussian blue settles out. 

(c) Jaife's reaction. — Treat 5 cc. of the solution 
with a saturated solution of picric acid, and make it 
alkaline with i cc. of a 10 per cent NaOH. The solution 
immediately becomes a deep red. 

ExPT. 80. Xanthine and hypoxanthine (Salkowski). 
— "Dissolve 50 grams of meat extract in 500 cc. of water 
in a flask, and after the addition of 75-100 cc. of nitric 
acid (1.2 sp. gr.), to destroy substances which hinder 
the precipitation of the xanthine bases by silver nitrate, 
heat on the sand-bath until the solution has cleared up, 
which will require about three-quarters of an hour. 
After cooling make strongly alkaline with ammonia, 
filter from the phosphates which separate, and add an 
ammoniacal solution of 2.5 grams of silver nitrate in 
about 100 cc. of water. The precipitate, which consists 
for the most part of hypoxanthine silver, besides a little 
xanthine silver, is then collected on a filter and washed 
a few times with water. 

"The separation of these two xanthine bases is ac- 
complished by converting them into the silver nitrate 
compounds. These compounds conduct themselves dif- 
ferently towards nitric acid. The hypoxanthine silver 
nitrate compound is very difficultly soluble in nitric acid ; 
the xanthine silver nitrate compound is far more readily 
soluble. The following is the best method of procedure : 



54 Food and Nutrition 

"Put the still moist precipitate into a flask and pour 
over it a mixture of lOO cc. of nitric acid and lOO cc. 
of water; add i gram of urea, heat just to boiling, and 
let cool. The hypoxanthine silver contained in the pre- 
cipitate is converted into hypoxanthine silver nitrate, 
which remains partly undissolved and partly goes into 
solution, but separates out of the solution again on cool- 
ing. The addition of the urea is to prevent the formation 
of nitrous acid, which might decompose the xanthine 
bases. Filter the hypoxanthine silver nitrate off after 
a few hours, and wash until the wash water no longer 
reacts strongly acid. Let the filtrate (without the wash 
water) stand till next day and filter (without working 
up the precipitate, which is a mixture of hypoxanthine 
silver nitrate and xanthine silver nitrate). The filtrate 
is used to show the presence of xanthine. The hypo- 
xanthine silver nitrate is examined under the microscope 
(fine needles frequently grouped in the form of stars). 

"(a) Pour upon a small portion of the substance on 
a porcelain crucible cover a few drops of strong or 
fuming nitric acid, and evaporate cautiously to dryness 
over a small flame. A lemon-yellow residue results, 
which takes on an orange color when moistened, after 
cooling, with caustic soda solution. If a drop of water 
be then added, a yellow solution results, and this when 
evaporated again leaves an orange residue (distinction 
from the murexide reaction for uric acid). 

"{h) Pour upon a small portion of the substance in 
a dish a httle pure nitric acid of the specific gravity 1.2, 
and evaporate on the water-bath to dryness. The 



Laboratory Manual 55 

residue is scarcely perceptibly colored. On the addition 
of caustic soda it becomes pale yellow (distinction from 
xanthine and guanine, which under these conditions give 
the xanthine reaction). Hypoxanthine is further dis- 
tinguished by its solubility in ammonia and the insolu- 
bility of its compound with silver nitrate in nitric acid, 
and also by the crystal form of this compound. 

"(r) The filtrate from hypoxanthine silver nitrate 
contains xanthine, as previously stated, but only in small 
quantity. Make it alkaline with ammonia (or, in order 
to save ammonia, neutralize the greater part of the acid 
with soda or lime and then make alkaline With ammonia). 
Xanthine silver precipitates in brown or reddish flakes. 
These are filtered off, washed, suspended in water, some 
drops of ammonia added, heated, treated with a few 
drops of ammonium sulphide, shaken thoroughly, filtered 
from the silver sulphide, and evaporated (or the precip- 
itate may also be decomposed with hydrochloric acid and 
xanthine hydrochloride obtained on evaporation). Very 
frequently the silver sulphide passes through the filter; 
we then evaporate to dryness and extract the residue 
with boiling water. The xanthine thus obtained is usu- 
ally not quite pure and the quantity is very small. It 
suffices, however, for the xanthine test as wxll as for 
the so-called Weidel's reaction. 

"(ff) Xanthine test. — Dissolve the residue or half 
of it in nitric acid and evaporate cautiously to dryness 
on a crucible cover over a small flame. A lemon-yellow 
residue results, which becomes intensely red on moisten- 
ing with caustic soda, and on further heating purplish 



56 Food and Nutrition 

red. Add a few drops of water and warm; a yellow 
solution results, which again gives a red residue on 
evaporation (distinction from the murexide reaction for 
uric acid). 

"(^) The so-called WeideVs reaction. — Dissolve half 
of the xanthine obtained in bromine water, warming 
gently, evaporate the solution on the water-bath to dry- 
ness, and invert the dish over another which contains 
some ammonia. The residue becomes red." 

Questions 

1. In what ways is salt used in the preparation of 
meat? 

2. If you wish rare roast beef, what should be the 
maximum interior temperature of the roast? 

3. What are the factors in producing flavor in 
preparation of meat? 

4. What inferences concerning making of soup do 
you draw from Dr. Grindley's investigations in meat? 

5. Discuss the subject of basting of meats. 

6. What protein substances are drawn out in cold 
water extracts of meat? 

7. Do the substances extracted by hot and cold 
water differ in character or quantity ? What is the chief 
loss in cooking meat? How is the percentage of loss 
of water influenced by method of cooking? 

8. Discuss in a general way the changes which 
take place in meat during cold storage. 



WHEAT, FLOUR, AND BREAD 

References: Jago. 
Allen. 

Leffmann and Beam. 
Osborne. The Proteins of the Wheat 

Kernel. 
Osborne and Voorhees. The Proteids of 
the Wheat Kernel. Journal of Amer. 
Chem. Soc, Vol. XV, No. 6. 
Division of Chemistry. Bulletin No. 13, 

Pt. 9. 
Office of Experiment Stations, Bulletins 

Nos. 52, 67, 85, loi, 126, 143, 156. 
South Dakota Exp. Sta., Bulletin No. 82. 
U. S. Dept. of Agriculture, Office of Sec- 
retary, Circular No. 13. 
ExPT. 81. Examine wheat kernel. Examine sections 
of wheat kernel under microscope and make drawings. 
ExPT. 82. Review physical tests of flour which you 
have made with low grade and high grade flours from 
winter and spring wheat. 

ExPT. 83. Make "doughing tests" with three differ- 
ent kinds of flour as follows: Mix in an evaporating 
dish with a glass rod or spatula 45 grams of flour and 
a measured quantity of water, enough to make stiff 
dough. Repeat the experiment with the other flours, 
using water enough to make doughs of the same con- 

57 



58 Food and Nutrition 

sistency as No. i. Observe the cubic centimeters of 
water used in each case and estimate the amount of 
water to "dough" i pound of each of the flours. 

ExPT. 84. Preparation of gluten. — 45 grams flour; 
water to make a stiff dough. Let it stand one hour. 
Put in cheese cloth and wash in stream of water. For 
large amount of flour (as i pound) better to lay cloth 
over sieve and wash. The gluten remains on the cheese 
cloth. If gluten is to be baked, put in oven immediately. 

{a) Treat a small portion of this gluten with 10 per 
cent sodium chloride solution. Does it dissolve the 
gluten? Grind some of the gluten in a mortar with 
alcohol (70 per cent by volume). Filter. Observe 
change in residue. Explain it. Test the filtrate as fol- 
lows: To one portion add distilled water. Boil one 
portion. Make biuret test on another portion. What 
proteins have you obtained? 

(&) Test two flours in the aleurometer (see Allen). 
For further work in separation of proteins of wheat 
flour see Osborne. 

Questions 

1. Compare the flours obtained from spring and 
winter wheats, giving characteristics of each kind. 

2. What dift'erence in manipulation do these flours 
require in the making of bread? 

3. Describe the changes which the different food 
principles undergo in the making of bread. 

4. Discuss the factors that produce sour bread. 

5. What qualities of bread are affected by the stage 
at which fermentation is arrested? 



Laboratory Manual 59 

6. What are the characteristics of a good macaroni 
flour? 

7. Discuss American macaroni. 

8. Describe the general structure of the wheat 
grain. 

9. From what part of the grain is the larger part 
of the flour obtained ? 

10. What do you understand by the term "aleurone 
layer"? 

11. Do "long "and "short" process bread differ in 
flavor, texture, or chemical composition? 

12. How can you distinguish gliadin and glutenin? 

13. In what way do these substances influence the 
bread-making qualities of a flour? 

14. Upon what do the bread-making qualities of a 
flour depend? 

15. Upon what does the nutritive value of a flour 
depend ? 



DIGESTION OF STARCH 

ExPT. 85. Prepare an iodine solution. Make a 
starch paste by boiling 30 grams of starch in 500 cc. 
of water. Boil until it becomes translucent, stirring 
thoroughly. Put 25 cc. of this paste into each of six 
bottles and stand five of them on the water-bath, to be 
kept at a temperature of 40° C. 

Number the bottles i, 2, 3, 4, 5, and 6, respectively. 
Place No. 5 in ice water. Weigh out carefully i deci- 
gram of pepsin for No. i, i decigram of pancreatin for 
No. 2, I decigram of amylopsin each for 3 and 5. Put 
a small amount of saliva in No. 6. 

Put these weighed quantities of the ferments into 
their respective bottles, and shake thoroughly. Notice 
that (4) is only for comparison. 

How does the ferment in each case affect the starch 
mass? In which does it liquefy most? Take five ordi- 
nary test-tubes and fill with water. Set them in a row, 
or number with a gummed label. Drop one small drop 
of iodine solution into each by means of a pipette, and 
shake to mix. 

When the enzymes have been at work for five min- 
utes, take a clean pipette and drop one drop of No. i 
into the first tube of iodine solution and put one drop 
of No. 2 into second iodine tube, and so on for each 
tube. Rinse the pipette each time. Then shake the 
iodine solutions. Test tube (6) every few seconds by 

60 



Laboratory Manual 6 1 

adding a drop of solution from (6) to a drop of iodine 
on a white plate. 

No. 4 will show what pure starch and iodine will 
give. How do the others compare? In which do you 
find the least change of color of the iodine? Allow to 
stand 5 minutes more and repeat the same, having re- 
plenished your test-tubes with iodine solution. What 
is the effect of temperature as shown by the ice water? 

After you have settled the question as to which of 
these ferments is most active in digesting starch, test 
the product of digestion to determine what change has 
taken place in the starch. Compare the action of saliva 
on starch paste and raw starch. What do you conclude ? 



DIGESTION OF PROTEINS 

ExPT. 86. Fibrin may be used in the following 
experiments instead of white of egg, also natural instead 
of artificial gastric and pancreatic juices. 
References: Howell. 

Hammarsten. 
Salkowski. 

Grindley. Bulletin No. 193, U. S. Dept. of 
Agriculture, Office of Experiment Stations. 
I. Make up solutions as follows: 

1. Dilute 6 cc. HCl (1.19 sp. gr.) to i liter. 

2. Add to solution (1)1 gram pepsin (or 2 grams). 

3. Add to 1,000 cc. water i gram pepsin. 

4. Add to 1,000 cc. I per cent NagCOg, i gram 
pepsin. 

5. 1,000 cc. I per cent Na2C03 + 2 to 3 grams pan- 
creatin. 

6. 2 to 3 grams pancreatin to i liter solution ( i ) . 

7. 2 grams amylopsin + 1,000 cc. i per cent 
NaoCOg. 

II. Prepare test-tubes or bottles as follows: 
Boil an egg hard and put the white through sieve. 
Use in following experiments: 

(a) In tube (a) put >4 gram white of egg and 20 cc. 
solution (2). 

(b) y2 gram white of egg in one piece + 20 cc. 
solution (2). 

62 



Laboratory Manual 63 

(0)^2 gram white of egg in 20 cc. solution (i). 

(d) Yz gram white of egg in 20 cc. solution (3). 

(^) /^ gram white of egg in 20 cc. solution (4). 

(/) V^ gram white of egg in 20 cc. solution (5) and 
thymol. 

(^) y^ gram white of egg in 20 cc. solution (6). 

(K) Yz gram white of egg in 20 cc. solution (7). 

(i) Yz gram yolk of egg in 20 cc. solution (5). 

(/) Prepare a second tube like (a) and put in a cool 
place. 

(^) Prepare a tube like (2) and add a few shreds 
of muscle fiber washed until white. 

(/) Prepare a tube like {i) and add washed muscle 
fiber. 

{m) Prepare a fifth tube containing 20 cc. solu- 
tion (5) and add washed muscle fiber. 

Place tubes thus prepared and labeled in warm oven 
at 40° C. Let the materials digest for 2 or 3 days and 
note carefully the results. At the end of 2 or 3 days 
test the contents of the tube for the products of digestion. 

Products of Protein Digestion 

(a) Peptic digestion. — Dilute 3 cc. of concentrated 
HCl to 500 cc. with distilled water, and add Y gram 
of soluble pepsin. Add this solution to the whites of 
four boiled eggs rubbed through a sieve. Allow to 
stand in a warm place, preferably at 40° C, and not 
above 56° C, with frequent shaking. After four days 
filter, and test a small portion of the solution for acid 
albuminates by just neutralizing with NaOH; filter out 



64 Food and Nutrition 

the acid albuminate and test the filtrate for proteoses 
and peptones with Millon's reagent. Neutralize the 
main bulk of the filtrate with NaoCOg and filter out the 
acid albuminate. Heat the filtrate to boiling. A pre- 
cipitate here is due to coagulable proteins. Filter and 
evaporate the filtrate to 200 cc. on the water-bath. Sat- 
urate the solution with solid (NH4)2S04 and filter out 
the albumose precipitated. The precipitate of the albu- 
mose is complete only after long standing of a completely 
saturated solution. 

Make tests for acid albuminate in precipitate and for 
peptone in filtrate. 

{h) Pancreatic digestion of egg white. — Boil four 
eggs hard and rub the whites through a sieve. Put 
them in a stoppered flask with 500 cc. of i per cent 
NagCOg solution containing 2 or 3 grams of pancreatin 
(commercial). Use a little thymol or chloroform to 
prevent putrefaction. The mixture is digested at 40° C. 
for about 72 hours, then the content of the flask is made 
faintly acid with acetic acid and boiled. Filter and 
evaporate the filtrate to a thin sirup. Set in a cool 
place until crystals of tyrosin appear. Examine some 
of the crystals under the microscope. Decant through 
muslin (reserve filtrate for separation of leucin) and 
wash with water. These crystals may be used for the 
following experiments. (If crystals of leucin have 
separated also, heat material cautiously in a little water 
and filter. The leucin is dissolved.) 

I. Put a few crystals of tyrosin in a test-tube with 
a little water, add a few drops of Millon's reagent, and 



Laboratory Manual 6$ 

heat gently. The Hquid, especially on standing, turns 
deep red. 

2. Piria's test {Hawk). — "Warm a little tyrosin on 
a watch-glass on a boiling water-bath for 20 minutes 
with 3-5 drops of concentrated H2SO4. Tyrosin-sul- 
phuric acid is formed in the process. Now add CaCOg 
in substance slowly, with stirring, until the reaction of 
the solution is no longer acid. Filter, concentrate the 
filtrate, and add to it a few drops (avoid an excess) of 
very dilute neutral ferric chloride. A purple or violet 
color, due to the formation of the ferric salt of tyrosin- 
sulphuric acid, is produced. This is one of the most 
satisfactory tests for the identification of tyrosin." 

Leucin. — The solution decanted from the tyrosin is 
evaporated further on the water-bath, and on standing 
crystals of leucin form on the surface. Examine under 
the microscope. Compare with tyrosin. For further 
experiments see Hawk. 



DIGESTION OF FAT 

ExPT. 87. Digestion of fat (or action of pancreatin 
on fats). — Free a fresh pancreas (pig or beef) from fat. 
Cut up with the scissors. Then grind a few grams with 
fine sand in a mortar to a paste. Add water until it is 
a creamy consistency. Melt some butter in a test-tube 
and let curd settle. Mix a few grams with an equal 
volume of pancreas paste. Add some chloroform water 
and keep at a temperature of 40° for several hours. 
Then test the reaction with rosolic acid. It is well to 
know the reaction of the mixture before it is put in the 
warm oven. 

"Litmus-Milk" test. — Into each of two test-tubes 
introduce 10 cc. of milk and a small amount of litmus 
powder. To the contents of one tube add 3 cc. of neutral 
pancreatic extract, and to the contents of the other tube 
add 3 cc. of water or of hoiled neutral pancreatic ex- 
tract. Keep the tubes at 40° C. and note any changes 
which may occur. What is the result, and how do you 
explain it? 

Questions 

1. In what forms are the carbohydrates of the food 
absorbed in the body? 

2. What are the bad effects of an excessive use of 
sugar in the diet ? 

3. What is saccharine? What is its place in the diet? 

66 



Laboratory Manual 6/ 

4. Under what circumstances are sugar and starch 
avoided in the diet? 

5. What protein foods would you use in the diet of 
children ? 

6. What objections are urged against a high protein 
diet? A low protein diet? 

7. Name some foods that yield purin bodies. 

8. What results are likely to follow an excessive 
use of fat in the diet? 

9. In what pathological condition is fat particularly 
prescribed ? 

10. Why is cows' milk modified in infant feeding? 



APPENDIX 

1. Constant temperature hath for gelatinization of 
starch. — Place about 15 cc. of a i per cent starch sus- 
pension in a test-tube inserted in a cork, which will serve 
as a stopper to the outside tube of an ordinary Beekmann 
boiling point apparatus. The outside tube may then be 
filled with any desired liquid of constant boiling point, 
and the inner tube suspended so as to be in contact with 
only the vapors of the boiling bath. The vapors are con- 
densed and run back into the tube from a side arm 
connected with a condenser. Samples of the starch may 
be removed from the inner tube periodically and exam- 
ined under the microscope. By using baths of various 
liquids boiling below 100° C, the effect of long-continued 
but low heat on starch granules may be easily observed. 

2. Egg albumin. — Cut up with scissors the v/hites 
of one dozen eggs, turn these into an equal volume of 
saturated (NH4)2S04, then filter (the precipitate is 
globulin). To the filtrate add acetic acid (10 per cent) 
until a permanent precipitate appears, then add i cc. 
acetic acid for each 100 cc. of solution. Stand over 
night; filter. Dry the precipitate and powder. 

3. Globulin. — Dissolve the globulin precipitate by 
adding water (the ammonium sulphate in the precipitate 
is sufficient to make a dilute salt solution). Reprecipi- 
tate by adding about an equal volume of alcohol. Filter, 
dry, powder. 

68 



Laboratory Manual 69 

4. Ovovitellin. — Shake the yolks of two eggs with 
200 cc. of ether and add 5 cc. of alcohol. Let the pre- 
cipitate settle and pour off the ether solution as com- 
pletely as possible, then add 100 cc. of a 15 per cent NaCl 
solution to the precipitate. On shaking, the precipitate 
dissolves in the salt solution, which is somewhat turbid. 
Put the liquid in a separatory funnel and shake it with 
an equal volume of ether. Separate the aqueous fluid 
from the ether and let it stand until the next day. If 
it is turbid shake it again with ether. Draw off the 
aqueous fluid again, measure it, and pour into it ten 
times its volume of water. Let this stand until next day 
and filter. Wash with water and then with alcohol. This 
precipitate is mixed with some lecithin. This may be 
removed by adding absolute alcohol to the precipitate 
and boiling in a flask on the water-bath. Filter, wash 
with alcohol, then with ether, then grind in a mortar and 
dry in a desiccator. A fine yellow powder is different 
from that of the first precipitate (see "A Laboratory 
Manual of Physiological Chemistry," by Salkowski, 
1904). 

5. Ovomucoid. — Add to whites of six eggs four 
times their volume of water, shake thoroughly, and pour 
into twice the volume of boiling water made very slightly 
acid by acetic acid. Boil briskly over free flame (granite 
pan) to thoroughly coagulate albumin and globulin. 
Filter and evaporate filtrate (which should give no pre- 
cipitate with mercuric chloride) on water-bath to about 
40 cc, pour into 100 cc. absolute alcohol, let stand until 
precipitate settles. Filter, wash with alcohol and ether, 
and dry. 



70 Food and Nutrition 

{a) Hydrolysis of ovomucoid. — To a small portion 
of the ovomucoid add locc. dilute HCl (lo per cent) 
and boil for five minutes, adding water as necessary. 
Add KOH to neutralize, and test the resulting solution 
with Fehling's reagent for reducing substances. If a 
precipitate does not appear at once allow the solution to 
stand until it becomes cool. If this method does not 
give satisfactory proof of a reducing substance, long- 
continued boiling with HCl as directed in the hydrolysis 
of mucin may be employed. 

According to Salkowski, nucleo-protein may be ob- 
tained as follows: 

6. Free the pancreas from fat. Heat 200 grams of 
finely chopped pancreas to boiling with i liter of water, 
keep boiling for ten minutes, filter, and add cautiously 
to the filtrate, while still warm, about 10-15 cc. of 
30 per cent acetic acid, until a fine, flocculent precipitate 
begins to settle. If the precipitate does not settle well 
it is advisable to heat again. Filter, wash with water, 
remove the precipitate from the filter, grind it with 50 cc. 
of absolute alcohol, filter, treat the precipitate with about 
50 cc. of ether, filter next day, wash once with ether, 
and grind. The presence of phosphorus and pentose 
may be very easily shown in the somewhat impure 
nucleo-protein thus obtained. 

{a) To detect the phosphorus fuse a small quantity 
with the oxidizing mixture, and then proceed as under 
Casein. 

{h) To detect the pentose the phloroglucin and the 
orcin tests may be used. 



Laboratory Manual yi 

Phloroglucin test. — Pour on a very small quan- 
tity of the substance in a few cubic centimeters of 
hydrochloric acid, add a little phloroglucin, and heat to 
boiling — cherry-red color, then turbidity. Let cool some- 
what, shake with an equal volume of amyl alcohol, and 
examine this with the spectroscope — absorption band be- 
tween D and E. 

Orcin test. — Instead of phloroglucin take a few 
orcin crystals and proceed in the same way — reddish 
blue color, then precipitation of a blue pigment. The 
amyl alcohol turns red and after some time emerald 
green. Examine with the spectroscope — absorption band 
between C and D. 

y. Separation of proteose and peptones according to 
Hawk. — "Place 50 cc. of proteose-peptone solution in 
an evaporating dish or casserole, and half saturate it with 
(NH4)2S04 solution, which may be accomplished by 
adding an equal volume of saturated {']>[Y{^)^SO^ solu- 
tion. At this point note the appearance of a precipitate 
of the primary proteoses (protoproteose and heteropro- 
teose). Now heat the half-saturated solution and its 
suspended precipitate to boiling, and saturate the solution 
with solid (NH4)2SO^. At full saturation the secondary 
proteoses (deuteroproteoses) are precipitated. The pep- 
tones remain in solution. 

"Proceed as follows with the precipitate of proteoses: 
Collect the sticky precipitate on a rubber-tipped stirring 
rod, or remove it by means of a watch-glass to a small 
evaporating dish, and dissolve it in a little water. To 
remove the (NH4)2SO^, which adhered to the precipitate 



72 Food and Nutrition 

and is now in solution, add BaCOg, boil, and filter off 
the precipitate of BaSO^. Concentrate the proteose 
solution to a small volume and make the following tests : 

"(a) Biuret test. 

''(b) Precipitation by HNO3. — What would a pre- 
cipitate at this point indicate? 

"(c) Precipitation by trichloracetic acid. — This pre- 
cipitate dissolves on heating and returns on cooling. 

"(d) Precipitation by picric acid. — This precipitate 
also disappears on heating and returns on cooling. 

"(e) Precipitation by potassio-mer curie iodide and 
HCl. 

"(f) Coagulation test. — Boil a little in a test-tube. 
Does it coagulate? 

"(g) Acetic acid and potassium ferrocyanide test. 

"The solution containing the peptones should be 
cooled and filtered, and the (NH4)2S04 in solution re- 
moved by boiling with BaCOg. After filtering off the 
BaS04 precipitate, concentrate the peptone filtrate to a 
small volume and repeat the tests as given under the 
proteose solution. In the biuret test the solution should 
be made very strongly alkaline with solid KOH." 



BIBLIOGRAPHY 

Allen: Commercial Organic Analysis, Vol. I (3d ed.). Phila- 
delphia, 1898. Vol. II, Pt. I (3d ed.). Philadelphia, 1901. 
Vol. IV (2d ed.). Philadelphia, 1898. 

Beddard^ Hill, Ed kins, and McCleod: Practical Physiology. 
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73 



74 Food and Nutrition 

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